The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.

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The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5' end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2-linked-DNA breaks, which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc. Here, we describe a flow cytometry-based method that allows a rapid assessment of drug-induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug-induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology. © 2017 by John Wiley & Sons, Inc.